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Biomedical subjects

Koichi Kato

Publications and source records attributed to Koichi Kato.

At least 55 records · Page 3Linked to original sources
PubMed2005

Remembering a nuclear accident in Japan: did it trigger flashbulb memories?

Flashbulb memories are vivid memories of the details surrounding the discovery of an emotional event. We investigated whether the nuclear accident that occurred in Japan in 1999 produced flashbulb memories among people who lived near the accident site. A questionnaire was distributed twice (approximately 3 weeks after the accident and 1 year later) to (1) the residents of the communities surrounding the accident site, (2) the students at a university near the accident site, and (3) the students at two universities far away from the accident site. Flashbulb memory holders were defined as those individuals who showed consistent memories between test and retest. The results indicated that only a small percentage of participants formed flashbulb memories. Further, no age-related decline was found. Flashbulb memories were distinguished by perfect or near perfect scores on four attributes: source, place, activity, and people. The results also indicated that the ratings on emotional reactions, personal consequentiality, and surprise did not differentiate between the flashbulb and non-flashbulb memory holders. In contrast, the flashbulb memory holders reported rehearsing more than the non-flashbulb memory holders. These results supported the notion that flashbulb memories are formed through rehearsal rather than at encoding. However, it is also possible that rehearsal was a result of the flashbulb memory.

Adult↗
PubMed2005

Molecular mechanism of ubiquitin recognition by GGA3 GAT domain.

GGA (Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding) proteins, which constitute a family of clathrin coat adaptor proteins, have recently been shown to be involved in the ubiquitin-dependent sorting of receptors, through the interaction between the C-terminal three-helix-bundle of the GAT (GGA and Tom1) domain (C-GAT) and ubiquitin. We report here the crystal structure of human GGA3 C-GAT in complex with ubiquitin. A hydrophobic patch on C-GAT helices alpha1 and alpha2 forms a binding site for the hydrophobic Ile44 surface of ubiquitin. Two distinct orientations of ubiquitin Arg42 determine the shape and the charge distribution of ubiquitin Ile44 surface, leading to two different binding modes. Biochemical and NMR data strongly suggest another hydrophobic binding site on C-GAT helices alpha2 and alpha3, opposite to the first binding site, also binds ubiquitin although weakly. The double-sided ubiquitin binding provides the GAT domain with higher efficiency in recognizing ubiquitinated receptors for lysosomal receptor degradation.

ADP-Ribosylation Factors↗
PubMed2005

A thin carboxymethyl cellulose culture substrate for the cellulase-induced harvesting of an endothelial cell sheet.

Engineered tissues constructed with two-dimensionally organized cells provide promising parts for reconstructing damaged tissues. Here we propose a new method for fabricating a 2D sheet made of an endothelial cell monolayer. First a culture substrate was prepared by treating the glass surface with an amine-terminated organosilicon derivative, followed by the covalent attachment of a thin carboxymethyl cellulose (CMC) layer. Fibronectin was immobilized onto the CMC-coated surface to promote cell adhesion. These surfaces were characterized step by step by means of contact angle measurement and X-ray photoelectron spectroscopy. Porcine aortic endothelial cells adhered to the culture substrate and consequently formed a confluent monolayer. When the substrate-cell composite was immersed in a cellulase solution, a cell sheet was spontaneously detached from the substrate due to enzymatic digestion of the CMC layer. The cell-cell connections were well preserved in the cell sheet, even after detachment from the substrate, most likely due to the fact that cellulase is harmless to mammalian cells. The cell sheet could be transferred to other culture dish with the aid of a hydrophilic membrane support, retaining the proliferation activity of the cells. The results obtained in this study demonstrate that cellulase treatment of the CMC layer is a rational and efficient method for obtaining a 2D cell sheet.

Animals↗
PubMed2005

Therapeutic neovascularization using cord blood-derived endothelial progenitor cells for diabetic neuropathy.

Diabetic neuropathy is based on the impairment of nerve blood flow and the metabolic disorder. Although the vasodilating agents and anticoagulants improve nerve function and symptoms in diabetic neuropathy, more effective treatments are needed. Because endothelial progenitor cells (EPCs) have been identified in adult human peripheral blood, many studies have shown that transplantation of EPCs improves circulation to ischemic tissues. In this study, we have demonstrated that therapeutic neovascularization using human umbilical cord blood-derived EPCs reversed diabetic neuropathy. EPCs were isolated and expanded on day 7 of culture from cord blood mononuclear cells. Unilateral intramuscular injection of EPCs into hindlimb skeletal muscles significantly ameliorated impaired sciatic motor nerve conduction velocity and sciatic nerve blood flow in the EPC-injected side of streptozotocin-induced diabetic nude rats compared with the saline-injected side of diabetic nude rats. Histological study revealed an increased number of microvessels in hindlimb skeletal muscles in the EPC-injected side of diabetic rats. These findings suggest that transplantation of EPCs from cord blood may be a useful treatment for diabetic neuropathy.

Animals↗
PubMed2004

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.

Animals↗
PubMed2004

Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity.

Recently, it became clear that aminoglycoside antibiotics affect protein-protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as alpha1-antitrypsin and alpha-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured alpha-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.

Animals↗
PubMed2004

Immunohistochemical analysis of oxidative DNA damage in arsenic-related human skin samples from arsenic-contaminated area of China.

The appearance of 8-oxo-2'-deoxyguanosine (8-oxodG) was examined immunohistochemically using an 8-oxodG-monoclonal antibody in 28 cases of arsenic-related human skin tumors and in 20 cases of arsenic-unrelated human skin cancer to determine if the induction of oxidative stress participates in skin tumorigenesis caused by arsenics. The rate of 8-oxodG-positive was significantly higher in arsenic-related human skin cancer (28 of 28, 100%) than in arsenic-unrelated human skin cancer (3 of 20, 15%, P<0.01 by Chi2 test). Moreover, in all the arsenic-related skin samples, 8-oxodG was detected not only in tumor tissues but also in keratosis and normal tissues. These results suggest that the induction of oxidative stress may play an important role in arsenic carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2004

Recombinant curculin heterodimer exhibits taste-modifying and sweet-tasting activities.

Curculin from Curculigo latifolia is a unique sweet protein that exhibits both sweet-tasting and taste-modifying activities. We isolated a gene that encodes a novel protein highly homologous to curculin. Using cDNAs of the previously known curculin (designated as curculin1) and the novel curculin isoform (curculin2), we produced a panel of homodimeric and heterodimeric recombinant curculins by Escherichia coli expression systems. It was revealed that sweet-tasting and taste-modifying activities were exhibited solely by the heterodimer of curculin1 and curculin2.

Amino Acid Sequence↗
PubMed2004

The role of active arsenic species produced by metabolic reduction of dimethylarsinic acid in genotoxicity and tumorigenesis.

In recent research of arsenic carcinogenesis, many researchers have directed their attention to methylated metabolites of inorganic arsenics. Because of its high cytotoxicity and genotoxicity, trivalent dimethylated arsenic, which can be produced by the metabolic reduction of dimethylarsinic acid (DMA), has attracted considerable attention from the standpoint of arsenic carcinogenesis. In the present paper, we examined trivalent dimethylated arsenic and its further metabolites for their chemical properties and biological behavior such as genotoxicity and tumorigenicity. Our in vitro and in vivo experiments suggested that the formation of cis-thymine glycol in DNA was induced via the production of dimethylated arsenic peroxide by the reaction of trivalent dimethylated arsenic with molecular oxygen, but not via the production of common reactive oxygen species (ROS; superoxide, hydrogen peroxide, hydroxyl radical, etc.). Thus, dimethylated arsenic peroxide may be the main species responsible for the tumor promotion in skin tumorigenesis induced by exposure to DMA. Free radical species, such as dimethylarsenic radical [(CH(3))(2)As.] and dimethylarsenic peroxy radical [(CH(3))(2)AsOO.], that are produced by the reaction of molecular oxygen and dimethylarsine [(CH(3))(2)AsH], which is probably a further reductive metabolite of trivalent dimethylated arsenic, may be main agents for initiation in mouse lung tumorigenesis.

Animals↗
PubMed2004

Micropatterned, self-assembled monolayers for fabrication of transfected cell microarrays.

The aim of this study was to develop the cell microarray that allows efficient transfer of multiple genes into mammalian cells cultured on the microarray in a high-throughput fashion. A microarray was fabricated using a gold-coated glass plate having a micropatterned, self-assembled monolayer of alkanethiols carrying ionic and nonionic terminal groups. Plasmid DNA and a cationic lipid were loaded by alternate electrostatic adsorption to the microspots to obtain a plasmid DNA microarray. The loading and the release of lipid-DNA complex were studied by, respectively, the fluorescence staining of DNA and the imaging of the microarray with a surface plasmon resonance (SPR) apparatus. The transfection efficiency was evaluated by directly plating and culturing human embryonic kidney cells onto the microarray. The results demonstrated that cells which adhered to the DNA-loaded spots were transfected to express the encoded model proteins for several days. The chemistry of the monolayers and the number of alternate adsorption cycles had large effects on the efficiency of transfection. This may be explained from the availability of the lipid-DNA complex to the cells directly contacted. We conclude that the micropatterned, self-assembled monolayers greatly facilitate regionally defined loading of DNAs and expression of the encoded protein in mammalian cells.

Adsorption↗
PubMed2004

Structural basis of sugar-recognizing ubiquitin ligase.

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.

Amino Acid Sequence↗
PubMed2004

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.

ATP Binding Cassette Transporter 1↗
PubMed2004

Temperature-dependent isologous Fab-Fab interaction that mediates cryocrystallization of a monoclonal immunoglobulin G.

Mechanisms of the cryoprecipitation of cryoglobulins have long been studied but not fully understood. In the present study, we characterize a monoclonal immunoglobulin G1 (IgG1) cryoglobulin that forms sharp needle crystals upon cooling and therefore might be an ideal example for the elucidation of a mechanism of cryoprecipitation of cryoglobulins. Limited proteolysis by papain of this cryoglobulin resulted in disappearance of cryocrystallization. Interactions of the proteolytic fragments were examined by surface plasmon resonance measurements. A temperature-dependent isologous Fab-Fab interaction was observed for this cryoglobulin. Glycosylation profiling by use of the HPLC mapping technique revealed that cryoglobulin expresses homogeneous N-glycans in its Fc portion. These results suggest that (1) the temperature-dependent crystallization of the cryoglobulin was ascribed to its specific Fab-Fab interaction and (2) the unusual homogeneity of the Fc glycosylation promote the crystallization of this cryoglobulin.

Cryoglobulins↗
PubMed2004

Synthesis and characterization of stimuli-sensitive hydrogels having a different length of ethylene glycol chains carrying phosphate groups: loading and release of lysozyme.

In order to prepare a polymer matrix capable of loading protein at high density, anionic hydrogels were synthesized by copolymerizing a monomer carrying a pendant phosphate group, methacryloyl-polyoxyethyl phosphate, with N-isopropylacrylamide and N,N'-methylene-bis-acrylamide, and the stumuli-sensitivity of hydrogels was characterized. The number of repeating ethylene glycol units in the phosphate carrying monomer was 1, 2, 5 or 8. Lysozyme bearing a positive net charge was immobilized in the hydrogel through formation of polyelectrolyte complex. It was shown that the amount of complexed lysozyme reached to 1.7 g/g dry gel, when high content of a phosphate-carrying monomer with 5 ethylene glycol units was incorporated into a hydrogel. It was further found that lysozyme complexed with phosphate-carrying network could be released by immersion of the lysozyme/hydrogel composite in a phosphate buffer solution of pH 7.4 owing to the pH-sensitivity of the hydorgel but no lysozyme was released at pH 1.4. The initial rate of lysozyme release was varied depending on the length of the ethylene glycol chains possessed by a network polymer and the content of the phosphate-carrying monomer unit. Lysozyme released from the phosphate-carrying hydrogel was proved to retain enzymatic activity at a level similar to the activity of lysozyme, which had been kept in buffer solution.

Ethylene Glycol↗
PubMed2004

Transfection microarray of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified glass slide.

Cell-based microarrays are emerging as a tool for analyzing the functions of genes in cells. However, partly due to the difficulty of cell immobilization, the application of this method has been limited to adherent cells. We previously reported a method that rapidly and strongly attached living nonadherent cells to glass slides modified with a cell membrane anchoring reagent, designated a biocompatible anchor for membrane (BAM). Here we demonstrate that plasmid DNA deposited in a defined area on BAM-modified glass slides was transfected into nonadherent K562 cells immobilized on the DNA-deposited and BAM-modified slides. This method allowed the transfection of K562 cells not only with plasmid cDNA expression vectors but also with small interfering RNA (siRNA) at a defined location on the BAM-modified slides. We expect this methodology to greatly expand the scope of current cell microarray technology.

Biocompatible Materials↗
PubMed2004

[Designed molecular probes in brain research: molecular imaging of IP2 receptor in the human brain].

The study of the role of prostaglandins in living human brain requires a highly designed non-invasive molecular probe with a specific function in the central nervous system and high stability in an in vivo system in addition to the ability of blood-brain-barrier penetration. We succeeded in designing 15R-TIC, which binds with a novel prostacyclin receptor subtype (IP2) expressed specifically in the central nervous system. 15R-TIC exhibited a distinct nerve-protecting effect in both high oxygen and in vivo ischemic conditions. In addition, a rapid C-methylation reaction, developed in order to incorporate a short-lived 11C-positron nuclide into the molecule, realized the synthesis of 15R-[11C]TIC methyl ester with the radioactivity of 2.5 GBq. The molecular imaging was established for both monkey and human brains by intravenous injection of this positron emission tomography (PET) probe.

Animals↗